Target (protein/gene name):
Glyceraldehyde-3-phosphate dehydrogenase NCBI Gene # or RefSeq#:
Tc00.1047053503687.20 Gene ID: 3535978 Protein ID (NP or XP #) or Wolbachia#:
XP_806067 (or XP_806067.1) Organism: Trypanosoma cruzi Etiologic Risk Group (see link below): Risk Group 2 (RG2) - Parasitic Agents Background/Disease Information:
Chagas disease is a parasitic disease caused by the organism Trypanosoma cruzi that is prevalent throughout Central and South America. It is estimated that Chagas disease is
affecting 8 to 11 million people and it is predominant in rural and impoverished areas. Recently though, it has been spreading out from Latin America into North America, as evidenced by the increased risk for Chagas disease in south Texas. It is possible that there could already be hundreds of people infected in Texas but have yet to be diagnosed due to the difficulty in symptom recognition. Chagas disease has an acute phase and a chronic phase. In the acute phase, harsh symptoms are uncommon and often people are symptom free or experience only flu-like symptoms. Some symptoms during this phase can include “fever, fatigue, body aches, headache, rash, loss of appetite, diarrhea, and vomiting.” The chronic phase can remain silent for months to a lifetime depending on the specific case but people can develop two main kinds of complications: cardiac complications and intestinal complications. Treatment in the acute phase usually consists of antiparasitic treatment with the drug Benznidazole or its alternative Nifurtimox. Often when patients lapse into the chronic phase, symptomatic treatment is also recommended to deal with the cardiac or intestinal problems that can arise. Chagas disease is transmitted primarily by triatomine bugs, a blood-sucking insect vector that is often given the name “kissing bugs” because of their tendencies to feed on the faces of sleeping people. The bugs become infected themselves by biting an infected person or animal and then pass it on through their feces, which enters the body through cuts or mucous membranes. It can also be transmitted through blood transfusions, congenital fluids, organ transplants, and the consumption of food contaminated with the feces of triatomine bugs that have the disease. The population that is most susceptible is the geographic region where the triatomine bugs make their home due to their pivotal role as vectors. Once in the body, the T. cruzi parasite, known at this point as a metacyclic trypomastigotes, transform into amastigotes. The amastigotes then replicate by binary fission in the infected cells. They then transform into trypomastigotes and burst into the bloodstream where they can then infect other cells and repeat the previous process of replication. If a triatomine bug then bites a person, the bug will become infected and can pass it on to the next victim. In Central and South America, methods have been put into place to try to control the spread of Chagas disease. In an attempt to limit the amount of people exposed to the triatomine bugs, housing conditions have been improved and many have taken to spraying insecticide in hopes of killing infected bugs. Also, early detection protocols and more stringent blood donation screenings are being put into place.
Essentiality of this protein:
significant loss of fitness in bloodstream forms (3 days) from a study of an African trypanosome [alsford]
significant loss of fitness in bloodstream forms (6 days) from a study of an African trypanosome [alsford]
significant gain of fitness in procyclic forms from a study of an African trypanosome [alsford]
Structure Available (PDB or Homology model) -- PDB # or closest PDB entry if using homology model:
1QXS
1K3T -- Query Coverage (if not direct match): -- Max Ident (if not direct match):
Druggable Target (see Databases for this): 0.3 Current Inhibitors:
The following compounds were studied in this paper and determined to have inhibitory functions.
(8E)-C15:1_anacardic acid, (8E,11E)-C15:2-anacardic acid, (8E,11E,14E)-C15:3-anacardic acid, 2-(dodec-1-en-1-yl)-6-hydroxybenzoic acid, 2-pentadecyl-6-hydroxybenzoic acid, guajaverin, mangiferin, tiliroside Expression Information (has it been expressed in bacterial cells):
It has been expressed in bacterial cells, E. coli BL21 (DE3) using a pET-28a+) vector. Purification Method: It was purified using column chromatography methods with an Ni-NTA resign. The protein was tagged with a six histidine tag which facilitated this method. Image of protein (PyMol or etc):
length of your protein in Amino Acids: 371aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
40.2897kD Molar Extinction coefficient of your protein at 280 nm wavelength:
46410 (assuming all Cys residues are reduced)
Glyceraldehyde-3-phosphate dehydrogenase
NCBI Gene # or RefSeq#:
Tc00.1047053503687.20
Gene ID:
3535978
Protein ID (NP or XP #) or Wolbachia#:
XP_806067 (or XP_806067.1)
Organism:
Trypanosoma cruzi
Etiologic Risk Group (see link below):
Risk Group 2 (RG2) - Parasitic Agents
Background/Disease Information:
Chagas disease is a parasitic disease caused by the organism Trypanosoma cruzi that is prevalent throughout Central and South America. It is estimated that Chagas disease is
affecting 8 to 11 million people and it is predominant in rural and impoverished areas. Recently though, it has been spreading out from Latin America into North America, as evidenced by the increased risk for Chagas disease in south Texas. It is possible that there could already be hundreds of people infected in Texas but have yet to be diagnosed due to the difficulty in symptom recognition. Chagas disease has an acute phase and a chronic phase. In the acute phase, harsh symptoms are uncommon and often people are symptom free or experience only flu-like symptoms. Some symptoms during this phase can include “fever, fatigue, body aches, headache, rash, loss of appetite, diarrhea, and vomiting.” The chronic phase can remain silent for months to a lifetime depending on the specific case but people can develop two main kinds of complications: cardiac complications and intestinal complications. Treatment in the acute phase usually consists of antiparasitic treatment with the drug Benznidazole or its alternative Nifurtimox. Often when patients lapse into the chronic phase, symptomatic treatment is also recommended to deal with the cardiac or intestinal problems that can arise. Chagas disease is transmitted primarily by triatomine bugs, a blood-sucking insect vector that is often given the name “kissing bugs” because of their tendencies to feed on the faces of sleeping people. The bugs become infected themselves by biting an infected person or animal and then pass it on through their feces, which enters the body through cuts or mucous membranes. It can also be transmitted through blood transfusions, congenital fluids, organ transplants, and the consumption of food contaminated with the feces of triatomine bugs that have the disease. The population that is most susceptible is the geographic region where the triatomine bugs make their home due to their pivotal role as vectors. Once in the body, the T. cruzi parasite, known at this point as a metacyclic trypomastigotes, transform into amastigotes. The amastigotes then replicate by binary fission in the infected cells. They then transform into trypomastigotes and burst into the bloodstream where they can then infect other cells and repeat the previous process of replication. If a triatomine bug then bites a person, the bug will become infected and can pass it on to the next victim. In Central and South America, methods have been put into place to try to control the spread of Chagas disease. In an attempt to limit the amount of people exposed to the triatomine bugs, housing conditions have been improved and many have taken to spraying insecticide in hopes of killing infected bugs. Also, early detection protocols and more stringent blood donation screenings are being put into place.
Essentiality of this protein:
significant loss of fitness in bloodstream forms (3 days) from a study of an African trypanosome [alsford]
significant loss of fitness in bloodstream forms (6 days) from a study of an African trypanosome [alsford]
significant gain of fitness in procyclic forms from a study of an African trypanosome [alsford]
Complex of enzymes:
EC#:
1.2.1.12
Link to BRENDA EC# page:
http://www.tdrtargets.org/targets/view?gene_id=52306
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
The enzyme assay that will be performed will be spectrophotometric.
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/glyceraldehyde3phosdehydr1.Par.0001.File.tmp/glyceraldehyde3phosdehydr1.pdf
-- Reagent Costs
-- link to paper that contains assay information
http://www.sciencedirect.com.ezproxy.lib.utexas.edu/science/article/pii/S1046592810003232
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
1QXS
1K3T
-- Query Coverage (if not direct match):
-- Max Ident (if not direct match):
Druggable Target (see Databases for this):
0.3
Current Inhibitors:
The following compounds were studied in this paper and determined to have inhibitory functions.
(8E)-C15:1_anacardic acid, (8E,11E)-C15:2-anacardic acid, (8E,11E,14E)-C15:3-anacardic acid, 2-(dodec-1-en-1-yl)-6-hydroxybenzoic acid, 2-pentadecyl-6-hydroxybenzoic acid, guajaverin, mangiferin, tiliroside
Expression Information (has it been expressed in bacterial cells):
It has been expressed in bacterial cells, E. coli BL21 (DE3) using a pET-28a+) vector.
Purification Method:
It was purified using column chromatography methods with an Ni-NTA resign. The protein was tagged with a six histidine tag which facilitated this method.
Image of protein (PyMol or etc):
Amino Acid Sequence:
>gi|71407160|ref|XP_806067.1| glyceraldehyde-3-phosphate dehydrogenase [Trypanosoma cruzi strain CL Brener]
MPLLLLRWLRRTVLLFCHWRRVIRMEDVVLPVCVGINGFGPIGQAVLFSSFTDPLVSVVAINDASMSIDY
IAYLLRRESSLSAGDRASVLVVGEFICIQGSQKIRVSHKHDLVEIAWRDVGVQYVVECTGLSSTRERCWG
HVAGGAKGVIVAGQSADAPTLIAGANDEELKSACSVVCAGSPVAVALAPLIRLLHEQYGLEECSYTAIHG
MRPVELTAGRSKNPQDWRQTRVSIDNIVPYIDNGKKTMDKIFPGLVGRISGTAFQVPVKKGCAVDMLVRL
SQPVSKEMLDQALKEAASGRLNGVLSYSKEDLISCDCVPNGKLCYDATGSCSLRDGEAQKLLLWFDIDGG
YAKRLLSLVVLLHQMGPSDGM
length of your protein in Amino Acids:
371aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
40.2897kD
Molar Extinction coefficient of your protein at 280 nm wavelength:
46410 (assuming all Cys residues are reduced)
CDS Gene Sequence:
ATGCCGCTGCTGCTTCTGCGGTGGCTACGACGGACAGTGTTGCTTTTTTGCCATTGGAGA AGAGTGATAAGGATGGAGGATGTTGTCTTGCCGGTTTGTGTGGGCATTAATGGCTTTGGG CCAATTGGGCAGGCCGTTCTTTTTTCCTCGTTCACGGACCCTTTAGTGAGCGTGGTGGCG ATAAACGATGCTTCCATGAGTATTGACTACATTGCATACTTGCTGCGGCGCGAAAGCTCT CTTTCTGCAGGGGACAGAGCCTCAGTGTTGGTGGTTGGGGAGTTTATCTGCATCCAGGGT TCTCAGAAAATCCGTGTTTCACATAAGCATGATCTTGTGGAGATTGCATGGCGAGATGTC GGGGTGCAGTACGTGGTGGAGTGCACGGGACTAAGCTCCACGCGAGAACGCTGCTGGGGA CATGTTGCAGGAGGTGCCAAAGGCGTCATTGTTGCAGGACAGAGTGCGGACGCCCCAACT CTCATTGCTGGTGCAAATGACGAAGAATTGAAAAGTGCGTGTTCCGTTGTGTGTGCTGGC TCCCCTGTTGCAGTCGCCCTCGCTCCTCTCATTCGTTTGCTGCATGAGCAATACGGTCTA GAAGAGTGCTCCTACACCGCCATTCACGGTATGAGACCAGTGGAGCTTACAGCAGGAAGG TCTAAAAATCCGCAAGACTGGCGCCAAACAAGAGTGAGTATTGACAACATTGTTCCTTAT ATCGACAACGGGAAAAAGACTATGGATAAAATTTTCCCAGGTCTTGTGGGACGCATCTCA GGAACTGCGTTTCAGGTCCCCGTGAAAAAAGGATGTGCCGTGGACATGTTGGTCCGGTTG AGCCAGCCTGTCTCGAAGGAAATGTTGGATCAAGCTCTGAAGGAAGCTGCTTCAGGTCGA CTCAACGGAGTGCTTTCTTACTCAAAAGAGGACCTCATTAGTTGCGACTGTGTGCCAAAT GGTAAGCTGTGTTACGACGCAACAGGTTCCTGTTCTCTGCGTGATGGGGAGGCGCAAAAA CTACTGTTATGGTTTGATATTGATGGTGGTTACGCGAAGAGGCTTCTGTCATTAGTGGTG
CTTTTGCATCAGATGGGTCCCAGTGACGGGATGTAG
GC% Content for gene:
50.99%
CDS Gene Sequence (codon optimized) - after having done Primer Design:
ATGCCTCTGCTCCTGCTGCGTTGGCTGCGTCGTACTGTACTCCTCTTTTGCCACTGGCGC CGTGTAATCCGCATGGAAGACGTAGTTCTGCCAGTATGTGTAGGTATCAACGGTTTCGGT CCTATCGGTCAGGCGGTTCTGTTTTCTAGCTTTACCGATCCTCTGGTTTCTGTAGTTGCT ATTAACGACGCCTCTATGTCTATCGACTACATCGCGTACCTGCTCCGTCGCGAATCTTCT CTGTCTGCCGGTGACCGTGCGAGCGTTCTCGTAGTTGGTGAGTTTATTTGCATCCAGGGT TCTCAGAAAATCCGTGTTTCTCACAAACACGACCTGGTTGAAATCGCGTGGCGCGACGTT GGTGTTCAGTACGTTGTTGAATGCACCGGTCTCTCCTCTACGCGTGAACGTTGCTGGGGT CACGTGGCGGGTGGTGCTAAAGGTGTTATTGTTGCGGGTCAGTCTGCGGACGCCCCGACC CTGATCGCAGGTGCGAACGACGAAGAACTGAAATCTGCATGCTCTGTTGTTTGCGCCGGT TCCCCGGTAGCGGTTGCTCTGGCCCCACTGATCCGTCTCCTCCACGAGCAGTACGGTCTG GAAGAATGTTCTTACACCGCGATTCACGGTATGCGTCCGGTTGAACTGACCGCGGGTCGT TCTAAAAACCCGCAGGACTGGCGTCAGACCCGTGTGTCCATTGATAACATCGTTCCGTAC ATCGACAACGGTAAGAAGACCATGGACAAAATCTTCCCGGGTCTCGTCGGCCGTATTTCT GGTACTGCATTCCAGGTTCCGGTTAAGAAAGGTTGTGCGGTTGACATGCTGGTTCGTCTG TCTCAGCCGGTTTCTAAAGAAATGCTGGACCAGGCGCTGAAAGAAGCGGCGTCTGGTCGT CTGAACGGCGTTCTGTCTTACTCTAAGGAAGACCTGATCTCTTGCGACTGCGTTCCGAAC GGCAAACTGTGCTACGATGCGACCGGTTCTTGTTCTCTGCGTGATGGCGAAGCGCAAAAA CTGCTGCTGTGGTTCGACATTGACGGTGGTTACGCGAAACGCCTCCTCTCCCTGGTGGTT
CTGCTGCACCAAATGGGTCCGAGCGATGGTATGTAA
GC% Content for gene (codon optimized):
54.12%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
1 ATGCCTCTGCTCCTGCTGCGTTGGCTGCGTCGTAC 35
2 TTCCATGCGGATTACACGGCGCCAGTGGCAAAAGAGGAGTACAGTACGACGCAGCCAACG 60
3 CCGTGTAATCCGCATGGAAGACGTAGTTCTGCCAGTATGTGTAGGTATCAACGGTTTCGG 60
4 TCGGTAAAGCTAGAAAACAGAACCGCCTGACCGATAGGACCGAAACCGTTGATACCTACA 60
5 GTTCTGTTTTCTAGCTTTACCGATCCTCTGGTTTCTGTAGTTGCTATTAACGACGCCTCT 60
6 TTCGCGACGGAGCAGGTACGCGATGTAGTCGATAGACATAGAGGCGTCGTTAATAGCAAC 60
7 CCTGCTCCGTCGCGAATCTTCTCTGTCTGCCGGTGACCGTGCGAGCGTTCTCGTAGTTGG 60
8 AAACACGGATTTTCTGAGAACCCTGGATGCAAATAAACTCACCAACTACGAGAACGCTCG 60
9 GGTTCTCAGAAAATCCGTGTTTCTCACAAACACGACCTGGTTGAAATCGCGTGGCGCGAC 60
10 GCGTAGAGGAGAGACCGGTGCATTCAACAACGTACTGAACACCAACGTCGCGCCACGCGA 60
11 CCGGTCTCTCCTCTACGCGTGAACGTTGCTGGGGTCACGTGGCGGGTGGTGCTAAAGGTG 60
12 CGATCAGGGTCGGGGCGTCCGCAGACTGACCCGCAACAATAACACCTTTAGCACCACCCG 60
13 GCCCCGACCCTGATCGCAGGTGCGAACGACGAAGAACTGAAATCTGCATGCTCTGTTGTT 60
14 CAGTGGGGCCAGAGCAACCGCTACCGGGGAACCGGCGCAAACAACAGAGCATGCAGATTT 60
15 TTGCTCTGGCCCCACTGATCCGTCTCCTCCACGAGCAGTACGGTCTGGAAGAATGTTCTT 60
16 CGGTCAGTTCAACCGGACGCATACCGTGAATCGCGGTGTAAGAACATTCTTCCAGACCGT 60
17 GTCCGGTTGAACTGACCGCGGGTCGTTCTAAAAACCCGCAGGACTGGCGTCAGACCCGTG 60
18 CTTCTTACCGTTGTCGATGTACGGAACGATGTTATCAATGGACACACGGGTCTGACGCCA 60
19 GTACATCGACAACGGTAAGAAGACCATGGACAAAATCTTCCCGGGTCTCGTCGGCCGTAT 60
20 CGCACAACCTTTCTTAACCGGAACCTGGAATGCAGTACCAGAAATACGGCCGACGAGACC 60
21 CGGTTAAGAAAGGTTGTGCGGTTGACATGCTGGTTCGTCTGTCTCAGCCGGTTTCTAAAG 60
22 GACCAGACGCCGCTTCTTTCAGCGCCTGGTCCAGCATTTCTTTAGAAACCGGCTGAGACA 60
23 AGAAGCGGCGTCTGGTCGTCTGAACGGCGTTCTGTCTTACTCTAAGGAAGACCTGATCTC 60
24 CGTAGCACAGTTTGCCGTTCGGAACGCAGTCGCAAGAGATCAGGTCTTCCTTAGAGTAAG 60
25 ACGGCAAACTGTGCTACGATGCGACCGGTTCTTGTTCTCTGCGTGATGGCGAAGCGCAAA 60
26 CGTTTCGCGTAACCACCGTCAATGTCGAACCACAGCAGCAGTTTTTGCGCTTCGCCATCA 60
27 CGGTGGTTACGCGAAACGCCTCCTCTCCCTGGTGGTTCTGCTGCACCAAATGGGTCCGAG 60
28 TTACATACCATCGCTCGGACCCATTTGGTGC 31
Google Docs Primer Design Log File:
https://docs.google.com/file/d/0B4O2KqKh2q_-LU01ZmdrOG5rT00/edit
Primer design results for 'tail' primers (this is just 2 sequences):